The present invention relates generally to the field of biochemical assays for the presence of peroxidase or other peroxidatively active substances. More specifically, it relates to a stabilizer useful both to stabilize and to solubilize an indicator for subsequent use to detect peroxidase activity.
The enzyme peroxidase acts to catalyze the redox reaction transfer of hydrogen from a hydrogen donor to a hydrogen acceptor. The hydrogen donor, or indicator, may be a chromogen which exhibits color in the oxidized state, so that peroxidase activity may be detected by monitoring color change. Alternatively, the hydrogen donor may be chosen from the class of indicators which emit light or exhibit other detectable features in the oxidized state. The hydrogen acceptor is typically hydrogen peroxide or a related compound such as methyhydroperoxide or ethylhydroperoxide.
The detection of peroxidase and peroxidase-like activity has utility in a variety of medically related fields. In the area of cytochemistry, for example, the detection of such activity can be used to identify and monitor certain cell types. Peroxidatively active substances are present in a restricted set of cell types including myeloid leukocytes and granulcytes, erythrocytes, and certain neurons and secretory cells. Mitotic cells also exhibit peroxidase-like activity. Assays sensitive to peroxidase activity can be used for certain medical determinations based on cytological condition.
Furthermore, in recent years assays based on peroxidase activity have provided a powerful tool in the immunochemical field, to aid in the detection of certain types of proteins, for example. Peroxidase is readily extractable and available in quantity from horseradish root. An antibody specific to a particular protein may be conjugated with the extracted peroxidase. After allowing the antibody to react with the protein antigen, the protein-antibody-peroxidase complex may be detected by providing appropriate hydrogen donors and acceptors and monitoring the redox reaction. Such methodology has been used in assays for a wide variety of ligands including proteins, peptides, carbohydrates and any other immunologically active substance.
In one such approach, antinuclear antibodies may be detected in human serum through the use of the peroxidase redox reaction. HEP-2 cells are fixed onto a slide, and then the cells are contacted with a serum which may contain antinuclear antibodies. If present in the serum, the antinuclear antibodies will react with the HEP-2 cells. After washing off the excess serum, the HEP-2 cells bonded with antinuclear antibodies are then further reacted with a solution containing antihuman IgG antibodies conjugated with peroxidase enzyme. The antihuman IgG antibodies are specific to any antinuclear antibodies bonded to the HEP-2 cells so that the peroxidase enzyme will be present on the slide after washing excess solution away only if antinuclear antibodies were present in the serum. Finally, the reacted cells fixed to the slide are contacted with a chromogenic indicator in a buffered aqueous solution which also contains peroxide, so that the peroxidase-catalyzed redox reaction may occur. By this procedure, the HEP-2 cells will be colored only to the extent that antinuclear antibodies were present in the serum reacted with the HEP-2 cells. With relatively minor modifications to the preparation of the slide and attached cells, other antigen reactions, such as for DNA or chlomydiae may be detected.
In another entirely different technique, "dip-and-read" type reagent bearing strips are used to detect peroxidase activity. These strips comprise a porous insoluble matrix strip first impregnated with a suitable hydrogen donor and acceptor and then dried. When immersed in a solution containing peroxidase, the indicator is oxidized, typically containing peroxidase, the indicator is oxidized, typically changing color. The "dip-and-read" technique has the advantage of ease of usage but obviously cannot be used in tests involving cell coloring by wet chemistry on a microscope slide.
A continuing problem with the peroxidase methodology for both the wet chemical approach and the dip-and-read strip approach is the gradual but spontaneous oxidation of the indicator. Such deterioration proceeds even more readily in an aqueous or peroxide containing medium. So rapid is this deterioration that reagent solutions must be prepared fresh within about a day prior to use, resulting in an excessive waste of valuable preparation time and costly reagents if all of each batch of reagent is not utilized.
Additionally, certain frequently used and desirable indicators, notably 4-chloro-1-napthol and 3-amino-9-ethylcarbazole, are insoluble in water. Accordingly, these types of compounds must first be dissolved in an organic solvent such as alcohol or dimethylsulfoxide to effect solubilization of the indicator before further mixing with an aqueous medium. These reagent solutions must also be prepared immediately prior to use as the indicator may precipitate out of solution upon standing.
Methods have been developed to inhibit spontaneous oxidation of the indicator on a dip-and-read strip. For example, the physical separation of the reagents may retard deterioration. Such separation may be effected by successive impregnation of the test strip with an indicator solution, a polyvinylmethylacylamide solution and a peroxide solution, with drying between each step. The polyvinylmethacrylamide forms a protective colloid, effectively separating the two reagents by a physical wall or barrier. Similarly, the peroxide may be encapsulated within a colloidal material such as gelatin which also acts as a physical barrier. In dip-and-read strips to which only an indicator is bound, certain lower alkyl or alkylene derivatives or monoalcohols have the effect of increasing the indicator's stability.
While applicable to the dip-and-read technique, such a physical barrier stabilization approach is obviously inappropriate for use in wet chemical methods. Reagent instability and insolubility continue to present serious limitations on the convenience and desirability of wet chemical peroxidase-related assay techniques.
The limitations are particularly apparent when tests such as the antinuclear antibody test described above are to be performed in a doctor's office, a field clinic, or by relatively unskilled personnel. Where the testing is to be performed at a location where the demand for testing is so low that only one or a few test are performed each day, a major portion of the costly indicator reagent solution may be discarded because the spontaneous oxidation renders the solution unreliable after short storage periods. When relatively unskilled persons are involved in the testing, the need to dissolve the indicator in an organic liquid prior to mixing with an aqueous solution may be overly complex, and may result in mixing errors which can invalidate the test procedure. Further, if peroxide must be added at the time of use because the indicator solution is unstable in the presence of peroxide, further mixing errors may result. Thus, it would be highly desirable to have an indicator which is readily soluble in aqueous solution and is stable for a period of time, even in the presence of peroxide. Such a solution could be prepared by one person and then used without further modification by a technician to prepare specimens for analysis.
Accordingly, there exists a need for a stabilized indicator/peroxide solution which may be prepared from a stable reagent which is soluble in water. Ideally, such a solution would be stable for an extended period of time, even in the presence of peroxide. The present invention fulfills this need, and further provides related advantages.